Thisprotocolwillvisualizebothinternalizedreceptorsthatwereonceontheplasmamembraneandreceptorsinthebiosyntheticpathway.ThisprotocolwasspecificallywrittenforvisualizingagoNIST-inducedinternalizationofreceptors.

Materials:

WashSolution--TotalVolume2liters;ManiatusTBSwith1mMCaCl2,16gNaCl,6gTrisbase,0.4gKCl,0.294gCaCl2.2H2O,pH7.4withconc.HCl.

Blotto--TotalVolume10mL,0.3gdrymilk(3%),100ul10%TritonX-100(0.1%),100ul100mMCaCl2(1mM),500ul1MTris-ClpH7.5(50mM).

Fixative--approximately2mlpercoverslip-37%formaldehydediluted1:10withPBS(150mMNaCl,10mMNaPi,pH7.4)

Procedure:

TissueCulture

Platecellsonto18mmsquarecoverslips.Pretreatmentofcoverslipwithpolylysinehelpscellremainadheredtothecoverslip.Don"tletthecellsgettooconfluent,orelsetheentire"sheet"ofcellswillcomeoffthecoverslipsduringthewashes.

1.Usinga6welltissuecultureplate,add2mlofmediatoeachwell.

2.Equilibratemediainincubator(~30-45").

3.Addcoverslips.Equilibrateinincubator(~25min).NOTE:Itispreferabletoperformbothequilibrationsteps;however,itisnotnecessary.

4.Treatcellswithagonist.(typically5-25").

5.Quicklyaspiratemediaandaddfixative(~2ml/well).Fixfor15-20".

6.Wash3x.

7.Aspiratethefinalwash,takingcaretodrytheedgesofthecoverslipwell.AddBlottotoonlythecoverslip(~50uL)andincubate20minutes.NOTE:WeonlyapplyBlottodirectlyontothecoverslipinordertoreducethevolumerequiredtoprocesseachcoverslip.VARIATION:SomepeopletransferthecoversliptoasheetofParafilminatissueculturedishwithasmallamountofwetwhatmaninside.Theparafilmhelpstheantibodysolution"beadup"onthecoverslip,andthewetwhatmanpaperhelpspreventtheantibodyfromdryingup.

8.AspirateBlotto,andapplyprimaryAbonlytothecoverslip.Incubate45".DiluteantibodywithBlotto.Sometypicaldilutionsarelistedbelow:monoclonalM1mouseanti-Flagantibody(Sigma,Covance)(Ca+2sensitive)1:350-1:500;polyclonal12CA5mouseanti-HA(Roche)1:1000.

9.Wash3x.

10.SecondBlottoblockasabove,5".

11.AspirateBlotto,andapplysecondaryAb.Incubate20"inthedark.DiluteantibodywithBlotto.Cy3donkeyanti-mouse(JacksonImmunoresearch)1:500,FITCgoatanti-mouse1:500,TexasRedgoatanti-mouse1:500.

12.Wash3x.

13.Wetmountwithanti-fadesolution,i.e.VectaShield(VectorLabs)orCitifluor(TedPella).Putsmalldropofmountingmediaonglassslide.Useforcepstopickupcoverslipandaspirateanyexcessliquid.Placecoverslipcell-sidedownontoglassslides.Aspirateanyexcessmountingmediafromedgesofcoverslip.Sealedgeswithnailpolish.

References:

JeffreyL.Benovic(Editor).RegulationofGProtein-CoupledReceptorFunctionandExpression(ReceptorBiochemistryandMethodology).

EpitopeTagging,BasicLaboratoryMethods.PDFmanualfromRochewebsite.